GhMPK17, a Cotton Mitogen-Activated Protein Kinase,Is Involved in Plant Response to High Salinity and Osmotic Stresses and ABA Signaling

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. Cotton (Gossypium hirsutum) is the most important textile crop in the world, and often encounters abiotic stress during its growth seasons. In this study, a gene encoding a mitogen-activated protein kinase (MAPK) was isolated from cotton, and designated as GhMPK17. The open reading frame (ORF) of GhMPK17 gene is 1494 bp in length and encodes a protein with 497 amino acids. Quantitative RT-PCR analysis indicated that GhMPK17 expression was up-regulated in cotton under NaCl, mannitol and ABA treatments. The transgenic Arabidopsis plants expressing GhMPK17 gene showed higher seed germination, root elongation and cotyledon greening/expansion rates than those of the wild type on MS medium containing NaCl, mannitol and exogenous ABA, suggesting that overexpression of GhMPK17 in Arabidopsis increased plant ABA-insensitivity, and enhanced plant tolerance to salt and osmotic stresses. Furthermore, overexpression of GhMPK17 in Arabidopsis reduced H₂O₂ level and altered expression of ABA- and abiotic stress-related genes in the transgenic plants. Collectively, these data suggested that GhMPK17 gene may be involved in plant response to high salinity and osmotic stresses and ABA signaling.     

Identification of novel stress-regulated microRNAs from Oryza sativa L.

MicroRNAs (miRNAs) are a type of small non-coding RNA found in eukaryotes. They play a key role in gene expression by down-regulating gene expression and are involved in the environment stress response in plants. Although a large number of miRNAs have been identified from Arabidopsis, few studies have focused on Oryza sativa miRNAs, especially on stress-related miRNAs. Five cDNA libraries of small RNAs from rice seedlings treated with cold, dehydration, salinity, and abscisic acid (ABA), as well as wild-type seedlings, were constructed. Seven rice novel miRNAs were identified by Northern analysis, and their expression patterns under different stress treatments were determined. Results showed that the expression of several novel miRNAs was regulated by one or more stress treatments. Our identification of novel stress-related miRNAs in rice suggests that these miRNAs might be involved in rice stress response pathways.     

Loss-of-function mutation of EIN2 in Arabidopsis exaggerates oxidative stress induced by salinity

Accumulation of reactive oxygen species (ROS) causes oxidative stress under adverse environmental conditions, such as salinity. Ethylene decreases accumulation of ROS induced by salinity, but the mechanism is still unclear. To examine the interactions between salinity and ROS accumulation and the possible role of ethylene metabolism in regulation, we used mutant ein2-5 in Arabidopsis with loss of function in EIN2. The mutant is compared to the wild-type Col-0, completely insensitivity to ethylene at the morphological, physiological and molecular levels. The oxidative responses of the wild type and mutant to salinity were compared. Loss-of-function of EIN2 enhanced sensitivity to salinity, implying that EIN2 is required for plant response to salinity. Furthermore, salinity resulted in accumulation of large amounts of ROS in ein2-5 seedlings when compared with Col-0, suggesting that the loss-of-function of EIN2 exaggerates oxidative stress induced by salinity. Activities of the antioxidant enzymes SOD, POD and CAT decreased significantly in ein2-5 under salinity when compared with Col-0 plants. The expression profiles of the genes Fe-SOD, PODs and CAT1, which code for ROS scavenging enzymes were severely decreased in ein2-5 under salinity compared with Col-0, suggesting that EIN2 was involved in regulating expression of these genes. Taken together, our results demonstrate that loss-of-function of EIN2 increased oxidative stress induced by salinity and that EIN2 is involved in modulating ROS accumulation, at least in part, by decreasing activities of ROS-scavenging enzymes.     

Overexpression of a wheat phospholipase D gene,TaPLDα, enhances tolerance to drought and osmotic stress in Arabidopsis thaliana

Phospholipase D (PLD) is crucial for plant responses to stress and signal transduction, however, the regulatory mechanism of PLD in abiotic stress is not completely understood; especially, in crops. In this study, we isolated a gene, TaPLDα, from common wheat (Triticum aestivum L.). Analysis of the amino acid sequence of TaPLDα revealed a highly conserved C2 domain and two characteristic HKD motifs, which is similar to other known PLD family genes. Further characterization revealed that TaPLDα expressed differentially in various organs, such as roots, stems, leaves and spikelets of wheat. After treatment with abscisic acid (ABA), methyl jasmonate, dehydration, polyethylene glycol and NaCl, the expression of TaPLDα was up-regulated in shoots. Subsequently, we generated TaPLDα-overexpressing transgenic Arabidopsis lines under the control of the dexamethasone-inducible 35S promoter. The overexpression of TaPLDα in Arabidopsis resulted in significantly enhanced tolerance to drought, as shown by reduced chlorosis and leaf water loss, higher relative water content and lower relative electrolyte leakage than the wild type. Moreover, the TaPLDα-overexpressing plants exhibited longer roots in response to mannitol treatment. In addition, the seeds of TaPLDα-overexpressing plants showed hypersensitivity to ABA and osmotic stress. Under dehydration, the expression of several stress-related genes, RD29A, RD29B, KIN1 and RAB18, was up-regulated to a higher level in TaPLDα-overexpressing plants than in wild type. Taken together, our results indicated that TaPLDα can enhance tolerance to drought and osmotic stress in Arabidopsis and represents a potential candidate gene to enhance stress tolerance in crops.     

Overproduction of the Membrane-bound Receptor-like Protein Kinase 1, RPK1, Enhances Abiotic Stress Tolerance in Arabidopsis

RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H₂O₂-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.     

Overexpression of a Lotus corniculatus AP2/ERF transcription factor gene,LcERF080, enhances tolerance to salt stress in transgenic Arabidopsis

The APETALA2/ethylene-responsive element binding factors (AP2/ERF) play central roles in the stress response in plants. In this study, we identified and isolated a novel salt stress-related gene, LcERF080, that encodes an AP2/ERF protein in Lotus corniculatus cultivar Leo. LcERF080 was classified into the B-4 group of the ERF subfamily based on multiple sequence alignment and phylogenetic characterization. Expression of LcERF080 was strongly induced by salt, abscisic acid, 1-aminocyclopropane-1-carboxylic acid, methyl jasmonate, and salicylic acid stresses. Subcellular localization assay confirmed that LcERF080 is a nuclear protein. LcERF080 overexpression in Arabidopsis resulted in pleiotropic phenotypes with a higher seed germination rate and transgenic plants with enhanced tolerance to salt stress. Further, under stress conditions, the transgenic lines exhibited elevated levels of soluble sugars and proline as well as relative moisture contents but a lower malondialdehyde content than in control plants. The expression levels of hyperosmotic salinity response genes COR15A, RD22, and P5CS1 were found to be elevated in the LcERF080-overexpressing Arabidopsis plants compared to the wild-type plants. These results reveal that LcERF080 is involved in the responses of plants to salt stress.     

A Leucine-Rich Repeat Receptor-like Kinase from the Antarctic Moss <Emphasis Type="Italic">Pohlia nutans</Emphasis> Confers Salinity and ABA Stress Tolerance

Plant leucine-rich repeats receptor-like kinases (LRR-RLKs) play key roles in plant growth, development, and responses to environmental stresses. However, the functions of LRR-RLKs in bryophytes are still not well documented. Here, a putative LRR-RLK gene, PnLRR-RLK, was cloned and characterized from the Antarctic moss Pohlia nutans. Phylogenetic analysis revealed that PnLRR-RLK protein was clustered with the Arabidopsis thaliana LRR XI family proteins. Subcellular localization analysis of PnLRR-RLK revealed that it was mainly localized on plasma membrane. The expression of PnLRR-RLK was induced by mock high salinity, cold, drought, and exogenously supplied abscisic acid (ABA) and methyl jasmonate (MeJA). Meanwhile, the overexpression of PnLRR-RLK showed an increased tolerance of transgenic Arabidopsis to salt and ABA stresses than that of the wild type (WT) plants. Furthermore, the expression levels of several salt tolerance genes (AtHKT1, AtSOS3, AtP5CS1, and AtADH1) and an ABA negatively regulating gene AtABI1 were significantly increased in transgenic plants. Meanwhile, the expression levels of ABA biosynthesis genes (AtNCED3, AtABA1, and AtAAO3) and ABA early response genes (AtMYB2, AtRD22, AtRD29A, and AtDREB2A) were decreased in transgenic Arabidopsis after salt stress treatment. Therefore, these results suggested that PnLRR-RLK might involve in regulating salt stress-related and ABA-dependent signaling pathway, thereby contribute to the salinity tolerance of the Antarctic moss P. nutans.     

Two cotton Cys2/His2-type zinc-finger proteins, GhDi19-1 and GhDi19-2, are involved in plant response to salt/drought stress and abscisic acid signaling

Cotton (Gossypium hirsutum) often encounters abiotic stress such as drought and high salinity during its development, and its productivity is significantly limited by those adverse factors. To investigate the molecular adaptation mechanisms of this plant species to abiotic stress, we identified two genes encoding Di19-like Cys2/His2 zinc-finger proteins in cotton. GFP fluorescence assay demonstrated that GhDi19-1 and GhDi19-2 are two nuclear-localized proteins. Quantitative RT-PCR and Northern blot analyses revealed that mRNA accumulation of both GhDi19-1 and GhDi19-2 was significantly promoted by salinity and drought. Expression of GUS gene driven by the GhDi19-1 and GhDi19-2 promoters, respectively, was intensively induced in cotyledons under NaCl and mannitol stresses. Overexpression of GhDi19-1 and GhDi19-2 in Arabidopsis resulted in the seedlings displaying hypersensitivity to high salinity and abscisic acid (ABA). Seed germination and seedling growth of the transgenic Arabidopsis were dramatically inhibited by salinity and ABA, compared with wild type. In addition, expression levels of the ABA-responsive genes ABF3, ABF4, ABI5 and KIN1 were also remarkably altered in the transgenic plants under ABA treatment. Collectively, our results suggested that both GhDi19-1 and GhDi19-2 may be involved in response to salt/drought stress and ABA signaling during early stages of plant development.     

The Arabidopsis LOS5/ABA3 Locus Encodes a Molybdenum Cofactor Sulfurase and Modulates Cold Stress– and Osmotic Stress–Responsive Gene Expression

To understand low temperature and osmotic stress signaling in plants, we isolated and characterized two allelic Arabidopsis mutants, los5-1 and los5-2, which are impaired in gene induction by cold and osmotic stresses. Expression of RD29A-LUC (the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter) in response to cold and salt/drought is reduced in the los5 mutants, but the response to abscisic acid (ABA) remains unaltered. RNA gel blot analysis indicates that the los5 mutation reduces the induction of several stress-responsive genes by cold and severely diminishes or even completely blocks the induction of RD29A, COR15, COR47, RD22, and P5CS by osmotic stresses. los5 mutant plants are compromised in their tolerance to freezing, salt, or drought stress. los5 plants are ABA deficient, as indicated by increased transpirational water loss and reduced accumulation of ABA under drought stress in the mutant. A comparison with another ABA-deficient mutant, aba1, reveals that the impaired low-temperature gene regulation is specific to the los5 mutation. Genetic tests suggest that los5 is allelic to aba3. Map-based cloning reveals that LOS5/ABA3 encodes a molybdenum cofactor (MoCo) sulfurase. MoCo sulfurase catalyzes the generation of the sulfurylated form of MoCo, a cofactor required by aldehyde oxidase that functions in the last step of ABA biosynthesis in plants. The LOS5/ABA3 gene is expressed ubiquitously in different plant parts, and the expression level increases in response to drought, salt, or ABA treatment. Our results show that LOS5/ABA3 is a key regulator of ABA biosynthesis, stress-responsive gene expression, and stress tolerance.     

Ethylene improves Arabidopsis salt tolerance mainly via retaining K+ in shoots and roots rather than decreasing tissue Na+ content

Ethylene has been reported to play an essential role in the response of Arabidopsis to salinity and K⁺ deficiency. It was proposed that plant's ability to maintain potassium (K⁺) and minimize sodium (Na⁺) in tissues of salinity plants is critical for salt tolerance (ST). It is still unclear how ethylene modulates plant ion homeostasis under saline occasions. We employed Arabidopsis wild type (Col-0), ethylene insensitive mutants (ein2-5 and ein3-1) and constitutive triple response mutant (ctr1-1) plants to compare their phenotypic and physiological responses to salinity. Ethephon applied to plants could convert quickly to ethylene and here was applied exogenously to Col-0 seedlings to validate ethylene role in salt response. We showed that ethylene insensitivity in ein2-5 or ein3-1 plants increased Arabidopsis salt sensitivity than in Col-0. However, the salinity-induced adverse effects on Chlorophyll a/b, photosystem II function (Fv/Fm) and redox state were largely amended in the ctr1-1 than in Col-0 plants with the severe salinity. The compatible solute sucrose and antioxidant system were also up-regulated to improve ST in ctr1-1 plants. The ethephon obviously alleviated the salinity-induced restriction in root length. The subsequent analysis on the Na⁺ and K⁺ homeostasis found that ethylene could help plant retain higher shoot or root K⁺ nutrition in the short- or long-term salt-stressed plants. However, the ethylene did not significantly alter sodium buildup and water relation in the salt-stressed plants. Our observations confirmed the key role of ethylene in improved plant ST and highlighted the ethylene ability to retain K⁺, rather than decreasing Na⁺, in shoots and roots to improve Arabidopsis ST.     

Overexpression of Arabidopsis Molybdenum Cofactor Sulfurase Gene Confers Drought Tolerance in Maize (Zea mays L.)

Abscisic acid (ABA) is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5) in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO) activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L.) exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC) and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA) and H₂O₂ content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT) maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses.     

AtHSP17.8 overexpression in transgenic lettuce gives rise to dehydration and salt stress resistance phenotypes through modulation of ABA-mediated signaling

Key message

Transgenic Arabidopsis and lettuce plants overexpressing AtHSP17.8 showed ABA-hypersensitive but abiotic stress-resistant phenotypes. ABA treatment caused a dramatic induction of early ABA-responsive genes in AtHSP17.8 -overexpressing transgenic lettuce.

Abstract

Plant small heat shock proteins function as chaperones in protein folding. In addition, they are involved in responses to various abiotic stresses, such as dehydration, heat and high salinity in Arabidopsis. However, it remains elusive how they play a role in the abiotic stress responses at the molecular level. In this study, we provide evidence that Arabidopsis HSP17.8 (AtHSP17.8) positively regulates the abiotic stress responses by modulating abscisic acid (ABA) signaling in Arabidopsis, and also in lettuce, a heterologous plant when ectopically expressed. Overexpression of AtHSP17.8 in both Arabidopsis and lettuce leads to hypersensitivity to ABA and enhanced resistance to dehydration and high salinity stresses. Moreover, early ABA-responsive genes, ABI1, ABI5, NCED3, SNF4 and AREB2, were rapidly induced in AtHSP17.8-overexpressing transgenic Arabidopsis and lettuce. Based on these data, we propose that AtHSP17.8 plays a crucial role in abiotic stress responses by positively modulating ABA-mediated signaling in both Arabidopsis and lettuce. Moreover, our results suggest that stress-tolerant lettuce can be engineered using the genetic and molecular resources of Arabidopsis.     

Overexpression of VaCBF4, a Transcription Factor from Vitis amurensis, Improves Cold Tolerance Accompanying Increased Resistance to Drought and Salinity in Arabidopsis

Environmental stress has a great impact on fruit yield and quality in grapes. Understanding mechanisms underlying stress tolerance in plants is useful for grape breeding. Here, a CBF gene, designated VaCBF4, was identified in V. amurensis. The expression of VaCBF4 was induced by several abiotic stresses, including cold, exogenous abscisic acid (ABA), drought, salinity, and cold-drought conditions. A yeast one-hybrid assay demonstrated that VaCBF4 protein could bind to a conserved DRE cis-element, which contains the core sequence ACCGAC and regulates cold- and dehydration-responsive. Transgenic Arabidopsis seedlings overexpressing VaCBF4 showed enhanced tolerance to cold, drought, and salinity when compared with wild-type controls. LT₅₀, a chilling temperature required to cause 50 % electrolyte leakage in leaves, was 4 °C lower in transgenic Arabidopsis lines than that in non-cold-acclimated wild-type seedlings. Moreover, two stress-responsive genes, AtRD29A and AtCOR47, also showed higher levels of expression in the transgenic lines than in wild-type seedlings under normal growth condition. Taken together, all these results clearly indicate that VaCBF4 is involved in the response to abiotic stresses, and it may be a good candidate gene for genetic improvement to develop stress-tolerant varieties in grapes.     

Knockout of AtDjB1, a J‐domain protein from Arabidopsis thaliana,alters plant responses to osmotic stress and abscisic acid

AtDjB1 is a member of the Arabidopsis thaliana J‐protein family. AtDjB1 is targeted to the mitochondria and plays a crucial role in A. thaliana heat and oxidative stress resistance. Herein, the role of AtDjB1 in adapting to saline and drought stress was studied in A. thaliana. AtDjB1 expression was induced through salinity, dehydration and abscisic acid (ABA) in young seedlings. Reverse genetic analyses indicate that AtDjB1 is a negative regulator in plant osmotic stress tolerance. Further, AtDjB1 knockout mutant plants (atj1‐1) exhibited greater ABA sensitivity compared with the wild‐type (WT) plants and the mutant lines with a rescued AtDjB1 gene. AtDjB1 gene knockout also altered the expression of several ABA‐responsive genes, which suggests that AtDjB1 is involved in osmotic stress tolerance through its effects on ABA signaling pathways. Moreover, atj1‐1 plants exhibited higher glucose levels and greater glucose sensitivity in the post‐germination development stage. Applying glucose promoted an ABA response in seedlings, and the promotion was more evident in atj1‐1 than WT seedlings. Taken together, higher glucose levels in atj1‐1 plants are likely responsible for the greater ABA sensitivity and increased osmotic stress tolerance.     

Isolation and characterization of two sorbitol transporter gene promoters in micropropagated apple plants (Malus?×?domestica) regulated by drought stress

The role of polyol transporters in stress tolerance in plants have been elucidated by many studies. Sorbitol transporter genes MdSOT3, MdSOT4 and MdSOT5 in apple plants, which are important for sorbitol loading and unloading, are regulated by drought stress. To further confirm the role of sorbitol transporters in stress tolerance, the constructs harboring MdSOT3 and MdSOT5 genes were introduced into wild type Arabidopsis plants (Col-0) and the Arabidopsis transformed with MdSOT3 or MdSOT5 performed higher drought stress tolerance compared to WT. In order to further understand how sorbitol transporters are involved in drought tolerance in apple plants, upstream regions of sorbitol transporter genes were isolated from apple plant source leaves by Anchored PCR from genomic DNA obtained, and then were used to drive expression of the GUS reporter in tobacco plants. The results showed that the longest fragments of MdSOT3 and MdSOT5 promoters induced the highest GUS activity under drought stress conditions. Additionally, fragments of these promoters that contain cis-acting elements known to be involved in stress response also induced GUS activity under drought stress. Taken together, our data suggest that increased MdSOT3 and MdSOT5 activity, through cis-acting elements in the promoters of these genes, play important roles in imparting tolerance to drought in micropropagated apple plants.